The gene encoding the prostatic tumor suppressor PSP94 is a target for repression by the Polycomb group protein EZH2.

PSP94, for prostatic secretory protein of 94 amino acids, is secreted by the prostate gland and functions as a suppressor of tumor growth and metastasis. The expression of PSP94 is lost in advanced, hormone-refractory prostate cancer and this correlates with an increased expression of the Polycomb protein EZH2 (enhancer of zeste homolog 2), which represses transcription via trimethylation of histone H3 on Lys27 (H3K27). We show here that these events are causally related and that the MSMB gene, which encodes PSP94, is trimethylated on H3K27 in androgen-refractory, but not in androgen-sensitive prostate cancer cells. Chromatin immunoprecipitation experiments confirmed an association of EZH2 with the MSMB gene. The RNAi-mediated knockdown of EZH2 resulted in a loss of H3K27 trimethylation and an increased expression of the MSMB gene. Conversely, the overexpression of EZH2 was associated with a decreased expression of the MSMB gene. We also demonstrate that MSMB is additionally repressed in androgen-refractory prostate cancer cells by the hypoacetylation of histone H3K9 and the hypermethylation of a CpG island in the promoter region. Our data disclose a hitherto unexplored link between the putative oncogene EZH2 and the tumor suppressor PSP94, and show that MSMB is silenced by EZH2 in advanced prostate cancer cells.

Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS1 and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS1 gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2Val-19, which carries the equivalent of the human H-rasVal-12 oncogene, was grown under conditions in which RAS1 expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2Val-19 mutant was grown under conditions in which RAS1 is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2(Val-19) oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS(Val-19) gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2(Val-19) strain under conditions in which RAS1 is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2(Val-19) oncogene. Hence, the present results show that the RAS protein in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS protein in S. cerevisiae apparently act similarly to the Gs proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway.

Identification of sds22 as an inhibitory subunit of protein phosphatase-1 in rat liver nuclei.

sds22 was originally identified in yeast as a regulator of protein phosphatase-1 that is essential for the completion of mitosis. We show here that a structurally related mammalian polypeptide (41.6 kDa) is part of a 260-kDa species of protein phosphatase-1. This holoenzyme, designated PP-1N(sds22), could be immunoprecipitated with sds22 antibodies and was retained by microcystin-Sepharose. PP-1N(sds22) is a latent phosphatase, but its activity could be revealed by the proteolytic destruction of the noncatalytic subunit(s). PP-1N(sds22) accounted for only 5-10% of the total activity of PP-1 in rat liver nuclear extracts. A synthetic 22-mer peptide, corresponding to a leucine-rich repeat of sds22, specifically inhibited the catalytic subunit of PP-1, showing that at least part of the latency stems from the interaction of the sds22 repeat(s) with PP-1C.

Molecular cloning of a human polypeptide related to yeast sds22, a regulator of protein phosphatase-1.

sds22 is a regulatory polypeptide of protein phosphatase-1 that is required for the completion of mitosis in both fission and budding yeast. We report here the cDNA cloning of a human polypeptide that is 46% identical to yeast sds22. The human homolog of sds22 consists of 360 residues, has a calculated molecular mass of 41.6 kDa and shows a tandem array of 11 leucine-rich repeat structures of 22 residues. Northern analysis revealed a major transcript of 1.39 kb in all 8 investigated human tissues. sds22 was detected by western analysis in both the cytoplasm and the nucleus of rat liver cells as a polypeptide of 44 kDa.

Identification of MYPT1 and NIPP1 as subunits of protein phosphatase 1 in rat liver cytosol.

Various studies have provided evidence for the existence of spontaneously active cytosolic species of protein phosphatase 1, but these enzymes have never been purified and characterized. We have used chromatography on microcystin-Sepharose and Resource Q to purify cytosolic protein phosphatases from rat liver. Two of the isolated enzymes were identified by Western analysis and peptide sequencing as complexes of the catalytic subunit of protein phosphatase 1 and either the inhibitor NIPP1 or the myosin-binding subunit MYPT1, which reportedly is not present in chicken liver. In contrast, PCR cloning revealed the expression of two MYPT1 splice variants in rat liver.

The transcriptional repressor NIPP1 is an essential player in EZH2-mediated gene silencing.

EZH2 is a Polycomb group (PcG) protein that promotes the late-stage development of cancer by silencing a specific set of genes, at least in part through trimethylation of associated histone H3 on Lys 27 (H3K27). Nuclear inhibitor of protein phosphatase-1 (NIPP1) is a ubiquitously expressed transcriptional repressor that has binding sites for the EZH2 interactor EED. Here, we examine the contribution of NIPP1 to EZH2-mediated gene silencing. Studies on NIPP1-deficient cells disclose a widespread and essential role of NIPP1 in the trimethylation of H3K27 by EZH2, not only in the onset of this trimethylation during embryonic development, but also in the maintenance of this repressive mark in proliferating cells. Consistent with this notion, EZH2 and NIPP1 silence a common set of genes, as revealed by gene-expression profiling, and NIPP1 is associated with established Polycomb target genes and with genomic regions that are enriched in Polycomb targets. Furthermore, most NIPP1 target genes are trimethylated on H3K27 and the knockdown of either NIPP1 or EZH2 is often associated with a loss of this modification. Our data reveal that NIPP1 is required for the global trimethylation of H3K27 and is implicated in gene silencing by EZH2.

Characterization of a ribosomal inhibitory polypeptide of protein phosphatase-1 from rat liver.

About 4% of the spontaneous phosphorylase phosphatase activity in a rat liver extract was associated with the ribosomal fraction and stemmed from both protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A). However, after repeated washing, only PP-1 remained bound to the ribosomes. The activity of ribosome-associated PP-1 (PP-1R) was partially latent and could be increased 2-3-fold by incubation with trypsin and an additional 50% by incubation with low concentrations of exogenous type-1 catalytic subunit. In contrast, incubation of the ribosomal fraction with MgATP resulted in a 50% drop in the activity of PP-1R. We have purified from a ribosomal extract a basic polypeptide (pI > or = 10.5) of 23 kDa that potently inhibited PP-1. This ribosomal inhibitor of PP-1, termed RIPP-1, was at least 30-times less efficient in inhibiting other major Ser/Thr protein phosphatases (PP-2A, PP-2B and PP-2C). RIPP-1 was identified as a non-competitive inhibitor of PP-1 with a substrate-dependent potency. The lowest Ki (approximately 20 nM) was obtained with phosphorylase and myelin basic protein as substrates. Besides instantaneously inhibiting the type-1 catalytic subunit, RIPP-1 also converted the catalytic subunit in a time-dependent manner (t 1/2 = 45 min at 25 degrees C) into a less active conformation. Unlike the inhibition, this slow inactivation was not reversed by the removal of RIPP-1. We propose that RIPP-1 accounts, at least in part, for the latency of PP-1R.

Cyclic AMP and the stimulation of trehalase activity in the yeast Saccharomyces cerevisiae by carbon sources, nitrogen sources and inhibitors of protein synthesis.

Addition of glucose to acetate-grown cells of Saccharomyces cerevisiae caused a rapid transient increase in the cAMP level followed by a 10-fold, transient increase in the activity of trehalase. Ethidium bromide and acridine analogues inhibited both glucose-induced responses in a similar way, confirming the role of the cAMP signal as the second messenger in the sugar-induced activation of trehalase. When nitrogen sources or protein synthesis inhibitors were added after the transient glucose-induced increase in the trehalase activity, a rapid reactivation of trehalase occurred. In this case, however, there was only a very small increase in the cAMP level, which appeared to be insignificant. When the nitrogen source or the protein synthesis inhibitor was added together with glucose, the trehalase activity remained high for a much longer time also without a significant effect on the cAMP level. When a membrane depolarizing agent was added together with the glucose, both the trehalase activity and the cAMP level remained high. Reversibility experiments in which trehalase was activated to different degrees also showed that for high sugar-induced trehalase activation a high cAMP level is needed, while nitrogen sources stimulate trehalase activity without affecting cAMP levels. In cell extracts, both cAMP and cGMP were able to activate trehalase, the latter however only at 10-fold higher concentrations. The cGMP level in vivo was about 10-fold lower than the cAMP level and was not significantly affected by nitrogen sources or protein synthesis inhibitors. Hence, neither cAMP nor cGMP seem to be involved as the second messenger in the stimulating effect of nitrogen sources and protein synthesis inhibitors on trehalase activity in yeast. Since all other evidence obtained here and before strongly points to regulation of trehalase by a 'cAMP-dependent' protein kinase, we suggest that the presence of a nitrogen source in the growth medium of yeast induces the rapid synthesis of an alternative second messenger able to activate this or another protein kinase.

Inactivation of nuclear inhibitory polypeptides of protein phosphatase-1 (NIPP-1) by protein kinase A.

We have recently purified two potent and specific inhibitory polypeptides of protein phosphatase-1 from the particulate fraction of bovine thymus nuclei (Beullens, M., Van Eynde, A., Stalmans, W., and Bollen, M. (1992) J. Biol. Chem. 267, 16538-16544). Here it is reported that these inhibitors, termed NIPP-1a (18 kDa) and NIPP-1b (16 kDa), are excellent substrates (Km = 0.1 microM) for phosphorylation by protein kinase A on both Ser and Thr residues. Phosphorylation was temporally closely related with an activation of NIPP-1. Maximal phosphorylation by protein kinase A (1.5 mol of phosphate/mol of NIPP-1) caused an 8-fold increase in the concentration of NIPP-1 required for half-complete inhibition of the catalytic subunit of protein phosphatase-1, irrespective of the concentration of the phosphatase. Phosphorylation decreased the binding of NIPP-1 to immobilized protein phosphatase-1. NIPP-1 could be efficiently and completely reactivated by incubation with the catalytic subunit of protein phosphatase-2A. The type-1 catalytic subunit was much less effective, however, even when present in a molar excess to NIPP-1. Chromatography of a salt extract of the particulate nuclear fraction of Mono Q revealed three species of PP-1. One of these species, termed PP-1N alpha, contained NIPP-1 as a subunit and could be activated 6-fold by incubation with protein kinase A under phosphorylating conditions. This activation of PP-1N alpha is opposite to the known inhibition of cytoplasmic species of protein phosphatase-1 by protein kinase A.

Full activation of a nuclear species of protein phosphatase-1 by phosphorylation with protein kinase A and casein kinase-2.

Bovine thymus nuclei contain a species of protein phosphatase-1 (PP-1N alpha) that can be partially activated by phosphorylation of an associated inhibitory polypeptide, NIPP-1, with protein kinase A [Beullens, Van Eynde, Bollen and Stalmans (1993) J. Biol. Chem. 268, 13172-13177]. Here it is shown that PP-1N alpha can also be activated 4-fold by phosphorylation of NIPP-1 with casein kinase-2. The effects of protein kinase A and casein kinase-2 were additive, yielding an enzyme with an activity close to that of the free catalytic subunit. Casein kinase-2 introduced up to 1.2 phosphate groups into purified NIPP-1 on serine and threonine residues. This phosphorylation was associated with a 14-fold increase in the concentration of NIPP-1 required for 50% inhibition of the type-1 catalytic subunit. The kinase-mediated inactivation of NIPP-1 could be reversed by incubation with the catalytic subunit of protein phosphatase-2A.

The isolation of novel inhibitory polypeptides of protein phosphatase 1 from bovine thymus nuclei.

Nuclei from bovine thymus contain a high level of partially latent protein phosphatase 1 (PP-1). More than 90% of this PP-1 is associated with the insoluble chromatin/matrix fraction and can be extracted with 0.3 M NaCl. The salt extract also contains three heat- and acid-stable inhibitory proteins of PP-1 that can be resolved on Mono Q. We have purified two of these nuclear inhibitors of PP-1 (NIPP-1a and NIPP-1b) until homogeneity. They are acidic proteins (pI = 4.4) with a molecular mass of 18 kDa (NIPP-1a) and 16 kDa (NIPP-1b) on SDS-PAGE. Judged from the larger molecular mass that was deduced from gel filtration (35 kDa), NIPP-1a and NIPP-1b appear to be asymmetric or dimeric proteins. The nuclear inhibitors totally inhibited the phosphorylase phosphatase activity of PP-1, but even at a 250-fold higher concentration they did not affect the activities of the other major serine/threonine protein phosphatases (PP-2A, PP-2B, and PP-2C). NIPP-1a and NIPP-1b inhibited the catalytic subunit of PP-1 with an extrapolated Ki of about 1 pM, which is some three orders of magnitude better than the cytoplasmic proteins inhibitor 1/DARPP-32 and modulator. The nuclear inhibitors were not inactivated by incubation with protein phosphatases that inactivate inhibitor 1 and DARPP-32. Unlike modulator, they were not able to convert the catalytic subunit of PP-1 into a MgATP-dependent form. Remarkably, the extent of inhibition of PP-1 by NIPP-1b depended on the nature of the substrate. The phosphorylase phosphatase and casein phosphatase activities of PP-1 were completely blocked by NIPP-1b, whereas the dephosphorylation of basic proteins was either not at all inhibited (histone IIA) or only partially (myelin basic protein). These data may indicate that the acidic NIPP-1b is inactivated through complexation by basic proteins. Indeed, nonphosphorylated histone IIA antagonized the inhibitory effect of NIPP-1b on the casein phosphatase activity of PP-1. Our data show that the nucleus contains specific and potent inhibitory proteins of PP-1 that differ from earlier described cytoplasmic inhibitors. We suggest that these novel proteins may control the activity of nuclear PP-1 on its natural substrate(s).

Subunit structure and regulation of protein phosphatase-1 in rat liver nuclei.

The activity of protein phosphatase-1 in rat liver nuclei (PP-1N) was decreased by up to 97% by associated inhibitory polypeptides, depending on the assay and extraction conditions. These inhibitors were rapidly degraded by endogenous proteases, resulting in the accumulation of active heat-stable intermediates. Two major species of PP-1N could be differentiated by fractionation of a nuclear extract. PP-1NR111 contained, besides the delta-isoform of the catalytic subunit, an inhibitory polypeptide of 111 kDa. PP-1NR41 was found to be an inactive heterodimer between the delta-isoform of the catalytic subunit and NIPP-1, a nuclear inhibitor of PP-1, which in its undegraded form is heat labile and migrates during SDS-polyacrylamide gel electrophoresis as a polypeptide of 41 kDa. Native hepatic NIPP-1 displayed a reduced affinity for the catalytic subunit after phosphorylation by protein kinase A in vitro and after glucagon-induced phosphorylation in vivo.

The C-terminus of NIPP1 (nuclear inhibitor of protein phosphatase-1) contains a novel binding site for protein phosphatase-1 that is controlled by tyrosine phosphorylation and RNA binding.

Nuclear inhibitor of protein phosphatase-1 (NIPP1; 351 residues) is a nuclear RNA-binding protein that also contains in its central domain two contiguous sites of interaction with the catalytic subunit of protein phosphatase-1 (PP1(C)). We show here that mutation of these phosphatase-interaction sites did not completely abolish the ability of NIPP1 to bind and inhibit PP1(C). This could be accounted for by an additional inhibitory phosphatase-binding site in the C-terminal region (residues 311-351), with an inhibitory core corresponding to residues 331-337. Following mutation of all three PP1(C)-binding sites in the central and C-terminal domains, NIPP1 no longer interacted with PP1(C). Remarkably, while both NIPP1 domains inhibited the phosphorylase phosphatase activity of PP1(C) independently, mutation of either domain completely abolished the ability of NIPP1 to inhibit the dephosphorylation of myelin basic protein. The inhibitory potency of the C-terminal site of NIPP1 was decreased by phosphorylation of Tyr-335 and by the addition of RNA. Tyr-335 could be phosphorylated by tyrosine kinase Lyn, but only in the presence of RNA. In conclusion, NIPP1 contains two phosphatase-binding domains that function co-operatively but which are controlled independently. Our data are in agreement with a shared-site model for the interaction of PP1(C) with its regulatory subunits.

Smooth flexible versus active tapered shaft design using NiTi rotary instruments.

AIM: The aim of this study was to evaluate the influence of a smooth flexible versus active tapered shaft design on canal preparation by NiTi rotary techniques. METHODOLOGY: A XMCT-scanner (SkyScan 1072) and developed software (Bergmans et al. 2001) were used to nondestructively analyze the mesial canals of 10 extracted mandibular molars in 3D with a spatial resolution of 30 microm. Specimens (n = 10 per group) were scanned before (PRE) and after (POST) preparation using Lightspeed (smooth flexible) or GT-rotary (active tapered) files. Numerical values for volumes, dentine removal (net) transportation and centring ability were obtained in addition to a visual inspection on canal aberrations. Data were analyzed by Shapiro Wilk test, multiway factorial anova, Tukey-Kramer test, Wilcoxon test and t-test. RESULTS: Results indicated that the active tapered shaft removed significantly more dentine in the middle to apical portion of the root compared to the smooth flexible design. Both groups demonstrated some straightening, but no significant differences were found with respect to instrument types. However, absolute values for net transportation and centering ratio were small and no canal aberrations could be found. CONCLUSIONS: The smooth flexible shaft design did not improve the morphological characteristics of canal preparation by NiTi rotary instruments when compared with the active tapered design. Therefore, system selection should be based upon other criteria.

Progressive versus constant tapered shaft design using NiTi rotary instruments.

AIM: To evaluate the influence of a progressive versus constant tapered shaft design on canal preparation by NiTi rotary techniques. METHODOLOGY: A XMCT-scanner and custom-made software were used to nondestructively analyse the mesial canals of 10 extracted mandibular molars in 3D with a spatial resolution of 12.5 microm. Specimens (n = 10 per group) were scanned before and after preparation using ProTaper (progressive tapered) or K3 (constant tapered) files. Numerical values for volume, curvature, dentine removal and centring ratio were obtained in addition to a visual inspection for canal aberrations. Data were analysed by (multiway factorial) anova, Wilcoxon tests and t-tests. RESULTS: The volume of total dentine removal (mean +/- SD) was 1.21 +/- 0.66 mm(3) (ProTaper) and 1.06 +/- 0.23 mm(3) (K3) (P > 0.05), and the amount of dentine removal at all separate horizontal regions examined was comparable for both groups. The mean linear dentine removal (transportation) was in the range of 8-212 microm (ProTaper) and 4-187 microm (K3). The resultant centring ratio varied from 0.01 to 0.24 (ProTaper) and from 0.01 to 0.17 (K3), whilst different straightening patterns were observed. A centre displacement towards the furcation coronally was most pronounced for the ProTaper group whereas a centre displacement towards the outer side of the curvature more apically was only observed for the K3 group. No severe canal aberrations were found. CONCLUSIONS: The progressive tapered shaft design of the ProTaper instrument was less influenced by the mid-root curvature than the constant tapered design of the K3 instrument thereby providing a good centred apical preparation. However, ProTaper instruments tended to transport towards the furcation in the coronal region.

Properties and phosphorylation sites of baculovirus-expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1).

NIPP-1 is the RNA-binding subunit of a major species of protein phosphatase-1 in the nucleus. We have expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1) in Sf9 cells, using the baculovirus-expression system. The purified recombinant protein was a potent (Ki = 9.9 +/- 0.3 pM) and specific inhibitor of protein phosphatase-1 and was stoichiometrically phosphorylated by protein kinases A and CK2. At physiological ionic strength, phosphorylation by these protein kinases drastically decreased the inhibitory potency of free NIPP-1. Phosphorylation of NIPP-1 in a heterodimeric complex with the catalytic subunit of protein phosphatase-1 resulted in an activation of the holoenzyme without a release of NIPP-1. Sequencing and phosphoamino acid analysis of tryptic phosphopeptides enabled us to identify Ser178 and Ser199 as the phosphorylation sites of protein kinase A, whereas Thr161 and Ser204 were phosphorylated by protein kinase CK2. These residues all conform to consensus recognition sites for phosphorylation by protein kinases A or CK2 and are clustered near a RVXF sequence that has been identified as a motif that interacts with the catalytic subunit of protein phosphatase-1.

Structure and splice products of the human gene encoding sds22, a putative mitotic regulator of protein phosphatase-1.

sds22 is a regulatory subunit of protein phosphatase-1 that is required for the completion of mitosis in yeast. It consists largely of 11 tandem leucine-rich repeats of 22 residues that are expected to mediate interactions with other polypeptides, including protein phosphatase-1. In this paper, we report on the structure of the human gene encoding sds22, designated PPP1R7. This gene (33 kb) comprises 11 exons, but these do not coincide with the sequences encoding the leucine-rich repeats. Up to six splice variants can be generated by exon skipping and alternative polyadenylation, as revealed by expressed sequence tag database analysis, RT-PCR and Northern blot analysis. The sds22 transcripts are expected to encode four different polypeptides. sds22alpha1 corresponds to the variant cloned previously from human brain [Renouf et al. (1995) FEBS Lett. 375, 75-78]. Sds22beta1 is truncated within the ninth repeat and has a short and different C-terminus. Both variants also exist without the sequence corresponding to exon 2, and these are termed sds22alpha2 and sds22beta2. The 5'-flanking region of PPP1R7 contains two NF-Y-binding CCAAT boxes near the transcription start site and potential binding sites for the transcription factors c-Myb, Ik-2 and NF-1, which are conserved in the mouse gene.

Studies on the mechanism of the glucose-induced cAMP signal in glycolysis and glucose repression mutants of the yeast Saccharomyces cerevisiae.

When glucose is added to cells of the yeast Saccharomyces cerevisiae grown on non-fermentable carbon sources, a cAMP signal is induced which triggers a protein phosphorylation cascade. Addition of glucose or fructose to cells of a phosphoglucose isomerase mutant also induced the cAMP signal indicating that metabolization of the sugar beyond the sugar phosphate step is not necessary. Glucose 6-phosphate might stimulate the triggering reaction since induction with fructose shows a significant delay. Experiments with double and triple mutants in hexokinase 1, hexokinase 2 or glucokinase indicated that the presence of one of the three kinases was both necessary and enough for induction of the cAMP signal by glucose and the presence of one of the two hexokinases necessary and enough for induction by fructose. The product of the kinase reaction itself however does not appear to be the trigger of the reaction: when the increase in the level of glucose 6-phosphate and fructose 6-phosphate was measured as a function of time after addition of different glucose concentrations, no correlation was observed with the increase in the cAMP level. From the dependence of the cAMP increase on the external concentration of glucose, a rough estimate was obtained of the Km of the triggering reaction: about 25 mM. This value clearly fits with the Km of the low-affinity glucose carrier (about 20 mM) and differs by at least an order of magnitude from the Km values of the high-affinity glucose carrier and the three kinases. The present results situate the primary triggering reaction at the level of transport-associated phosphorylation. The main (= low-affinity) glucose carrier appears to be the receptor while association of the corresponding kinase is needed for induction of the signal. Since it is known that the presence of the kinases influences the characteristics of sugar transport, no definite conclusion can be given on whether the necessity of the kinases reflects the need for a certain type of transport or the need for phosphorylation of the sugar. The increase in the level of fructose 1,6-bisphosphate, on the other hand, correlated very well with the cAMP increase. However, it clearly lagged behind the cAMP increase, confirming the previously suggested importance of the cAMP signal for the stimulation of glycolytic flux at the level of phosphofructokinase 1.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: intracellular pH and the effect of membrane depolarizing compounds.

Addition of plasma membrane depolarizing agents, such as dinitrophenol (DNP) and azide, to cells of Saccharomyces cerevisiae under aerobic conditions, is known to cause an increase in the cAMP level within 15 s. We found that both compounds lowered the intracellular pH (measured by in vivo 32P-NMR) drastically within the same time period. Plasma membrane depolarization, however, was much slower: DNP and azide had no effect on the membrane potential during, respectively, the first 2 min and the first 10 min after addition. Apparently, the intracellular pH of yeast is much more sensitive to perturbation than the membrane potential. The effect of both compounds on the cAMP level was highly dependent on the extracellular pH: when the latter was raised, the effect disappeared completely between pH 6 and 7. A similar dependence on the extracellular pH was observed for the lowering of intracellular pH. Addition of organic acids, such as acetate and butyrate, at low pH and under aerobic conditions, also caused an immediate increase in the cAMP level and an immediate drop in the intracellular pH. These results suggest that agents such as DNP and azide do not raise the cAMP level in yeast cells because of their membrane depolarizing properties but because they lower the intracellular pH. Under anaerobic conditions, DNP, azide and organic acids were much less effective in increasing the cAMP level. Addition of a small amount of glucose, however, restored their capacity to enhance the cAMP level. This suggests that under anaerobic conditions and in the absence of glucose the ATP level is a limiting factor for cAMP synthesis.